In current research and development process involving process involving nucleic acid (DNA and RNA) amplifications, there is a need for rapid, accurate qualitative determination of the results from such experiments. Traditionally, such determinations require the preparation of gels, for example, agarose, for electrophoresis of amplified nucleic acids and the use of a nucleic acid intercalating agent, for example, ethidium bromide, which is fluorescent under ultraviolet light. Generally, agarose gels are cast by melting a measured amount of agarose in a buffer resulting in a transparent solution. The melted agarose is poured into a mold and allowed to harden. The mold includes sample wells for adding the nucleic acids to be analyzed. In order to detect the nucleic acid, ethidium bromide is included in the buffer used to prepare the gel or the gel is stained with ethidium bromide after electrophoresis. Samples of the nucleic acids to be analyzed are mixed with a loading buffer and deposited into the sample wells. The gel is then subjected to a certain applied voltage resulting in the lateral migration of the nucleic acid through the gel. This method requires a power supply and an apparatus for casting and running the gels. Additionally, an ultraviolet light box is required for detecting the nucleic acid, requiring the use of protective eye wear. Further, the use of ethidium bromide poses an health hazard as it is a carcinogen, a powerful mutagen and moderately toxic. The qualitative determination process, as described above, is time consuming, requires special precautions for the handling and disposing of ethidium bromide, requires the purchase of specialized equipment, poses a potential health hazard and requires specialized training in the use of the necessary equipment. The sensitivity of the method is low and since equipment is used many times over, the possibility of contamination is also present.
The introduction of nucleic acid probe tests based on hybridization into routine clinical laboratory procedures has been hindered by lack of sensitivity when compared with conventional culture techniques and immunoassays. To increase sensitivity, radioactive labels are used. The problem with the use of such radioactive labels is the laboratory management of radioisotopes and the disposal thereof. To some degree, non-isotopic labels are used to overcome this problem. However, non-isotopic methods do not possess adequate sensitivity and are cumbersome and laborious to run. In order to achieve the sensitivity needed for diagnostics, the incubation times of the current methodologies must be long, taking up to several hours or days. The current non-isotopic methodologies also employ many steps, requiring previous training and expertise.
The ability to amplify nucleic acids from clinical samples has greatly advanced nucleic acid probe technology, providing the sensitivity lacking in earlier versions of non-isotopic assays. The sensitivity afforded by oligonucleotide probe tests utilizing nucleic acid amplification now exceeds that of any other method. However, the method is still time consuming, requiring the use of specialized hybridization techniques and equipment, transfer to solid phases and specialized detection equipment.
The detection of amplified nucleic acids for clinical use largely relies on hybridization of the amplified product with a detection probe that is labeled with a variety of enzymes and luminescent reagents. U.S. Pat. No. 5,374,524 to Miller, describes a nucleic acid probe assay which combines nucleic acid amplification and solution hybridization using capture and reporter probes. These techniques require multiple reagents, several washing steps, and specialized equipment for detection of the target nucleic acid. Moreover, these techniques are labor intensive and require technicians with expertise in molecular biology.
The use of probes comprised of oligonucleotide sequences bound to microparticles is well known and illustrated in the prior art. The mechanism for attachment of oligonucleotides to microparticles in hybridization assays and for the purification of nucleic acids is also well known. European Patent No. 200 133 describes the attachment of oligonucleotides to water-insoluble particles less than 50 micrometers in diameter used in hybridization assays for the capture of target nucleotides. U.S. Pat. No. 5,387,510 to Wu, describes the use of oligonucleotide sequences covalently bound to microparticles as probes for capturing PCR amplified nucleic acids. U.S. Pat. No. 5,328,825 to Findlay also describes an oligonucleotide linked by way of a protein or carbohydrate to a water-insoluble particle. The oligonucleotide probe is covalently coupled to the microparticle or other solid support. The sensitivity and specificity of the four above-reference patents, each of which is specifically incorporated herein, is based on hybridization of the oligonucleotide probe to the target nucleic acid.
The use of incorporated non-radioactive labels into the amplification reactions for the detection of nucleic acids is also well known in the art. Nucleic acids modified with biotin (U.S. Pat. No. 4,687,732 to Ward et al.; European Patent No. 063879; both specifically incorporated herein), digoxin (European Patent No. 173251, specifically incorporated herein) and other haptens have also been used. For example, U.S. Pat. No. 5,344,757 to Graf, specifically incorporated herein, uses a nucleic acid probe containing at least one hapten as a label for hybridization with a complementary target nucleic acid bound to a solid membrane. The sensitivity and specificity of these assays is based on the incorporation of a single label in the amplification reaction which can be detected using an antibody specific to the label. The usual case involves an antibody conjugated to an enzyme. Furthermore, the addition of substrate generates a colorimetric or fluorescent change which can be detected with an instrument.
Still, the above-described approaches are labor intensive with many steps and washes; require special and costly equipment for the detection of the target nucleic acid; require trained staff; and take a several hours to complete. Several patents have issued which deal with automation of the processes of amplification and subsequent detection of the amplicon. These patents use specialized equipment and are still based on the principle of hybridization and immunoassay technology. For example, European Patent No. 320308, specifically incorporated herein, describes a system for detecting target nucleic acids amplified by the ligase chain reaction.
Automated approaches eliminate the need for specially trained personnel, however, the cost of the equipment is very high and the possibility of contamination still exits since many samples will be processed by the same equipment.
The use of bifunctional labels for detection of an amplified target sequence has been explored using the ligase chain reaction (European Patent No. 320308, supra). Upon completion of the amplification by the ligation process, double stranded DNA is formed with biotin bound at one end and fluorescein bound at the other end. The labeled nucleic acid serves as an analyte in a two site immunometric assay. That is, a microparticle coated with anti-fluorescein antibodies captures the ligated product; after a wash step, a second biotin specific antibody labeled with alkaline phosphatase binds to the biotin yielding a fluorescent signal when incubated with a suitable substrate. The signal can be read with a fluourmeter. This system is based on immunometric technology and requires specialized equipment for detection of the target nucleic acid.